Many enzymes that are useful in the synthesis of fine chemicals and pharmaceutical intermediates require co-factors such as NAD(H) or NADP(H), for example the bioconversion of ketones to alcohols (see M.-R. Kula, 1994, “Enzyme Catalyzed Reductions or Carbonyl Groups”, Proceedings of Chiral Europe, 27-33). However, it is prohibitively expensive to add the co-factor as needed in large-scale reactions. A method of regenerating the co-factor is required to make such syntheses economically feasible. This can often be accomplished by the use of a second enzyme capable of reducing the oxidized form of the co-factor, which is then utilized by the primary enzyme to complete the desired conversion.
Enzymatic regeneration has been accomplished through the use of glucose-6-phosphate dehydrogenase (C.-H. Wong and G. M. Whitesides, 1994, J. Am. Chem. Soc. 103:4890-4899), alcohol dehydrogenase (C.-H. Wong and G. M. Whitesides, 1982, J. Org. Chem. 47:2816-2818), and formate dehydrogenase (M.-R. Kula, 1994, “Enzyme Catalyzed Reductions or Carbonyl Groups”, Proceedings of Chiral Europe, 27-33; also see C.-H. Wong and G. M. Whitesides, 1994, “Enzymes in Organic Chemistry”, Pergamon Press, Elsevier Science Ltd., Oxford, UK).
FDH is the preferred enzyme for reactions requiring NADH as co-factor because it uses an inexpensive substrate, sodium formate, as a hydrogen source and releases only CO2 as a byproduct. This enzyme has been isolated from a number of methylotropic bacterial (Berezin et al., SU 543672) and yeast (Cordes et al., EP 86109675 and U.S. Pat. No. 4,879,234; Hollenberg et al., U.S. Pat. No. 5,389,525) strains for use in large-scale co-factor regeneration. In addition, the FDH enzyme has been isolated from P. pastoris strain NRRL-Y-7556 (C. T. Hou et al., 1982, Arch. Biochem. Biophys. 216:296-305).
Despite the great interest in the various forms of formate dehydrogenase, the isolation of the FDH gene and expression of the recombinant enzyme from Pichia pastoris has not been reported. Given the importance of FDH, it is highly desirable to have an additional source of the enzyme. Although use of the native organism to induce expression of FDH would require the use of methanol, a flammable and hazardous solvent, heterologous expression of the protein in a host such as Escherichia coli would allow inexpensive, high-level production of the enzyme while avoiding the requirement of methanol induction. In addition, recombinant production of the enzyme is expected to be a faster and less expensive process than purification. Accordingly, the present invention provides an novel recombinant formate dehydrogenase isolated from P. pastoris which can be used in various reactions, including those useful for synthesis of fine chemicals and pharmaceutical intermediates.